Development and In Vitro Evaluation of 2-Methoxyestradiol Loaded Polymeric Micelles for Enhancing Anticancer Activities in Prostate Cancer

Abstract

The present study aimed to formulate and optimize 2ME-loaded PMs (2ME-PMs) for enhancing the anticancer activity of 2ME in prostate cancer (PC). The 2ME-PMs were formulated using PEG-PLGA (PL), Tween 80 (TW80), and alpha-lipoic acid (ALA). The optimization was carried out using a Box-Behnken design with the PL, TW80, and ALA as the independent variables and particle size (PS) as the response. The formulation was optimized for the lowest possible PS, and the software suggested optimum formula with 100.282 mg, 2%, and 40 mg for PL, TW80, and ALA, respectively. The optimized PMs had spherical morphology with PS of 65.36 ± 2.2 nm, polydispersity index (PDI) of 0.273 ± 0.03, and entrapment efficiency of 65.23 ± 3.5%. The in vitro drug release was 76.3 ± 3.2% after 24 h. The cell line studies using PC-3 cells showed IC₅₀ values of 18.75 and 54.41 µmol for 2ME-PM and 2ME, respectively. The estimation of tumor biomarkers was also carried out. The tumor biomarkers caspase-9 (17.38 ± 1.42 ng/mL), tumor protein P53 (p53) (1050.0 ± 40.9 pg/mL), nitric oxide (NO) (0.693 ± 0.03 pg/mL), interleukin-1β (IL-1β) (25.84 ± 2.23 pg/mL), nuclear factor kappa B (NF-kB) (0.719 ± 0.07 pg/mL), interleukin-6 (IL-6) (2.53 ± 0.16 folds), and cyclooxygenase-2 (COX-2) (3.04 ± 0.5 folds) were determined for 2ME-PMs and the results showed that these values changed significantly compared to those of 2ME. Overall, the results showed that the formulation of 2ME to 2ME-PMs enhances the anticancer effect. The exploration of the combined advantages of PEG, PLGA, ALA, and PMs in cancer therapy and the delivery of 2ME is the major importance of this research work. PEG reduces the elimination of 2ME, PLGA enhances 2ME loading, ALA has an inherent apoptotic effect, and PMs can efficiently target tumor cells.

Keywords: 2-methoxyestradiol; cell viability assay; cytotoxicity; optimization; polymeric micelles; prostate cancer.

Figure 1

Results for particle size (PS) after carrying out design of experiments by Box-Behnken design for the formulation of 2-methoxyestradiol-loaded polymeric micelles (2ME-PMs) prepared with PEG-PLGA (PL), Tween 80 (TW80), and alpha-lipoic acid (ALA) as independent factors (a) standardized Pareto chart (b) main effects plot (c) contours of the response surface.

Figure 1

Results for particle size (PS) after carrying out design of experiments by Box-Behnken design for the formulation of 2-methoxyestradiol-loaded polymeric micelles (2ME-PMs) prepared with PEG-PLGA (PL), Tween 80 (TW80), and alpha-lipoic acid (ALA) as independent factors (a) standardized Pareto chart (b) main effects plot (c) contours of the response surface.

Figure 2

TEM image of optimized 2-methoxyestradiol-loaded polymeric micelles (2ME-PMs) prepared with the optimum formula of 100.282 mg, 2%, and 40 mg for PEG-PLGA (PL), Tween 80 (TW80), and alpha-lipoic acid (ALA), respectively.

Figure 3

In vitro release of 2-methoxyestradiol (2ME) from 2ME-loaded polymeric micelles (2ME-PMs) in phosphate-buffered saline (PBS) at 37 °C, pH 6.8 (500 mL) containing Tween 80 (0.5%) as a solubilizer by dialysis bag method.

Figure 4

Cell viability assay results of control, placebo, 2-methoxyestradiol (2ME), and 2ME-loaded polymeric micelles (2ME-PMs) following cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in PC-3 cells [Statistical inferences: @, p < 0.05, compared with control; †, p < 0.05, compared with placebo; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].

Figure 5

Apoptotic activity of samples by Annexin V-FITC and propidium iodide and flow cytometry (a) Dot plots showing PC-3 cells treated with control, 2-methoxyestradiol (2ME), and 2ME-loaded polymeric micelles (2ME-PMs) (b) Bar diagram showing a quantitative data of percent of cells in various stages of apoptosis. [Statistical inferences: @, p < 0.05, compared with control; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].

Figure 6

Histograms derived from cell cycle analysis of samples by Annexin V-FITC and propidium iodide and flow cytometry. The percent of cells observed in each phase was quantified after control, placebo, 2-methoxyestradiol (2ME), and 2ME-loaded polymeric micelle (2ME-PM) treatments in PC-3 cells. [Statistical inferences: @, p < 0.05, compared with control; †, p < 0.05, compared with placebo; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].

Figure 7

Mitochondrial membrane potential (MMP) activities of control, 2-methoxyestradiol (2ME), and 2ME-loaded polymeric micelles (2ME-PMs) in PC-3 cells determined using tetramethylrhodamine methyl ester probe and flow cytometry. The histograms represent the percentage loss of MPP after treatment with the samples. [Statistical inferences: @, p < 0.05, compared with control; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].

Figure 8

Effect of 2-methoxyestradiol (2ME) and 2ME-loaded polymeric micelles (2ME-PMs) on various biomarkers (a) Caspase-9 (b) tumor protein P53 (p53) (c) nitric oxide (NO) (d) tumor necrosis factor-alpha (TNF-α) (e) interleukin-1β (IL-1β) (f) nuclear factor kappa B (NF-kB) (g) interleukin-6 (IL-6) (h) cyclooxygenase-2 (COX-2) [Statistical inferences: @, p < 0.05, compared with control; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].

Figure 8

Effect of 2-methoxyestradiol (2ME) and 2ME-loaded polymeric micelles (2ME-PMs) on various biomarkers (a) Caspase-9 (b) tumor protein P53 (p53) (c) nitric oxide (NO) (d) tumor necrosis factor-alpha (TNF-α) (e) interleukin-1β (IL-1β) (f) nuclear factor kappa B (NF-kB) (g) interleukin-6 (IL-6) (h) cyclooxygenase-2 (COX-2) [Statistical inferences: @, p < 0.05, compared with control; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].

Figure 9

Bax (a) and Bcl-2 (b) gene expressions after treatment with control, placebo, 2-methoxyestradiol (2ME), and 2ME-loaded polymeric micelles (2ME-PMs). [Statistical inferences: @, p < 0.05, compared with control; †, p < 0.05, compared with placebo; #, p < 0.05, compared with 2ME; $, p < 0.05, compared with 2ME-PMs].